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57 Cards in this Set
- Front
- Back
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What does the nucleus tell us about a cell? |
-cells and nuclei vary in morphology= can determine which tissue came from |
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Nuclear structures |
1. nuclear membrane (envelope) 2. chromatin 3. nucleolus 4. nuclear pores |
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Function of nuclear structures |
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Nuclear import |
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Nuclear export |
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Nuclear membrane |
-2 membranes: 1. outer membrane --continuous w/ RER --ribosome studded 2. inner membrane --associates w/ chromatin (heterochromatin, interphase) |
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Nuclear pores |
-discontinuities in nuclear envelope -physical communication -3000-4000/cell -shape: --octagonal symmetry --donut shaped (70-100 nM diameter) --central channel (9 nM) --sometimes central granule EM: Cytoplasmic face: octagonal structure nucleoplasmic face: basket structure |
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Immunofluroescent detection of proteins |
-Abs labeled w/ fluorophore that bind to taarget protein -DAPI: stains DNA --> nucleus appears blue |
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Nuclear lamina |
-gives nucleus structure -composed of Lamins (A, B, C) --similar to intermediate filament proteins --form mesh-work of filaments, assembled in 2-D lattice beneath nuclear envelope --10-20 nm thick --interrupted by nuclear pores -Lamin Associated Proteins |
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Lamin Associated Proteins (LAPs) |
-Inner nuclear membrane proteins: --tether lamina to nuclear membrane --Ex: emerin -DNA binding proteins --tether chromatin to lamina --Ex: Lap2ß |
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Laminopathies
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-specific mutations in Lamin A --> predisposition to: (b/c of changes in gene expression) 1. Hutchinson-Gilford Progeria Syndrome (HGPS) 2. Dilated Cardiomyopathy 3. Familial partial lipodystrophy 4. Emery-Dreisfuss muscular dystrophy (EMD1) 5. Limb-girdle muscular dystrophy 6. Charcot-Marie Tooth disease 7. Mandibuloacral dysplasia **phenotype: disrupted nuclear structure= abnormal shape, disrupted chromatin attachment (not on edge of nucleus, concentrated in middle) |
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Hutchinson-Gilford Progeria Syndrome (HGPS) |
-Lamin A mutation--> short Lamin A protein w/ persistent farnesylation --> Lamin A permanently attached to nuclear envelope --> nuclear integrity severely compromised (abnormal shape vs. circle) -phenotype: premature aging -survival rarely beyond mid-teens |
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??Nuclear envelope= nuclear membrane + nuclear lamina |
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nuclear lamina and mitosis |
interphase: kinases phosphorylate lamins and nuclear-binding proteins--> prophase: polymer breaks down = lattice falls apart --> membrane breaks into small pieces --> dephosphorylation --> telophase --> fusion of nuclear envelope fragments --> late telophase : decondensing of chromosomes --> fusion of enveloped chromosomes --> interphase |
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Nuclear pore complex |
-125 MDa -nucleoporins: 50-100 proteins that compose complex -regulate passage of molecules bw/n nucleus and cytoplasm --small molecules (<50kDa) freelly diffuse --larger molecules (50 + kDa) active transport = export/imiport cargo |
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Nuclear import |
-Nuclear Localization Sequence (NLS): --basic aa rich sequence: Lys-Lys-Lys-Arg-Lys (optimal core) --essential and sufficient for nuclear import -discovered studying histone proteins -many different importins w/ different specificities |
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Nuclear export |
-discovered studying RNA -Nuclear Export Sequence (NES): --leucine-rich: Leu-X(1-2)-Leu-X(2-3)-Leu-X-Leu |
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Nuclear proteins/protein complexes exported |
1. small proteins that need actively excluded (freely difused in through pore but aren't wanted) 2. assembled ribosomal units (RNPs and proteins in RNP complexes) 3. large NLS-containing proteins that function in both nucleus and cytoplasm --contain NLS and NES --Ex: c-Abl |
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Ran |
-G-protein -regulates import/export -activity/conformation altered by GDP vs. GTP bound -GTPase that only works w/ presence of Ran GTPase activity factor present (Ran-GAP) -GTP hydrolysis by Ran provides directionality of nuclear transport -Ran-GEF: Ran exchange factor |
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Nuclear Import Steps |
1. Cytoplasm: Importin bunds NLS on cargo--> importin binds nuclear pore--> ipmortin & cargo enter nucleus 2. Nucleus: Ran-GTP binds importin --> importin affinity for cargo decreases --> cargo released --> Ran-GTP-importin move across nuclear pore to cytoplasm Ran Cycle: 3. Cytoplasm: Ran-GAP binds Ran-GTP --> hydrolyzed to Ran-GDP --> Ran-GDP can't bind importin --> Ran-GDP binds its receptor --> imported into nucleus 4. Nucleus: Ran-GDP bound by Ran-GEF --> GDP exchanged for GTP--> Ran-GTP |
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Nuclear Export Steps |
1. Nucleus: Ran-GTP binds exportin --> increased affinity for NES on cargo --> binds cargo --> Ran-GTP-exportin-cargo move through nuclear pore 2. Cytoplasm: Ran-GTP bound by Ran-GAP --> GTP hydrolyzed to Ran-GDP --> exportin releases cargo --> exportin moves back into nucleus (Ran-GDP undergoes Ran cycle) |
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RNA export |
(not Ran and GTP hydrolysis driven) -NXC1, NXT1: exportins, coat RNA -DBS5 helicase displaces exoprtins in cytoplasm, uses ATP, ATP hydrolysis drives rxn |
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Cyclin D1a vs D1b |
-D1b isoform: increased cancer risk --NES affected--> isn't exported from nucleus |
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Nucleolus |
-subnuclear body, not membrane bound= not organelle -rDNAA clustering, rRNA synthesis, ribosome assembly 3 areas distinguishable by EM: 1. associated (hetero)chromatin 2. fibrillar area: active rRNA synthesis (fuzzier looking) 3. granular are: ribosome assembly (granular looking) |
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Sub-nuclear bodies |
not membrane bound -immunofluoerescence detectable 1. speckles 2. cajal bodies 3. PML bodies |
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Speckles |
-0.2-0.5 um -RNA processing centers |
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Cajal bodies |
-0.15-1.5 um 0-10/nucleus -possible splicing centers, RNA processing, associate w/ nucleolus, telomerase transport |
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PML bodies |
-0.3-1.0 u -10-30/nucleus -protein repository (storage) |
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Does embedding DNA in chromatin change perception of its availability for info read-out? |
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How important is control of cell cycle? |
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How is analysis of nucleus useful in medical applications? |
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Chromatin |
-linear DNA nad associated proteins packaged as chromatin in order to fit in nucleus: DNA=1 meter, nucleus=5-7 um -enables: --orderly replication/segregation --additional regulatory mechanism of transcription |
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nucleosome |
ev. conserved structure basic unit of chromatin (chromatin fiber is extended array of linked nucleosomes = beads on a string) DNA + histone proteins: -H2a, H2b dimerize -> dimers w/ dimer -H3, H4 dimerize -> dimers w/ dimer ----> form octamer -H1 linker bw/n octamers -120-140 bps/octamer -50 bp linker DNA |
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chromatin higher order structure |
1. nucleosome array (beads on a string) 2. supercoiling --> 30 nm wide fiber 3. 30 nm wide fibers folded into looped domains so fit in nucleus = euchromatin-like 4. transitional folding state approaching mitosis= heterochromatin-like 5. condensins package into fully condensed state= metaphase chromosome, visible in light micro |
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Covalent modifications of euchromatin |
DNA: CpG hypomethylated Histones: hyperacetylated, H3 lys 9 unmethylated |
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Covalent modifications of heterochromatin |
DNA: CpG hypermethylated Histones: deacetylated, H3 lys 9 methylated |
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the histone code |
-most common modifications: (occur on N-terminal histone tail) methylation, acetylation, phosphorylation -reading histone code: --protein complexes which regulate gene expression recognize the pattern of histone modifications --> determines if gene will be turned on/off |
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epigenetics |
-niheritance of histone modifications -clinical importance: 1. patterns of inheritance 2. cancer (ex: p16 repression) 3. stem cells: nuclear reprogrammingrequires altering epigenetics to change gene expression patern |
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Mitotic Cell cycle |
1. M phase: mitosis, cytokinesis 2. interphase: a. G1- Gap phase 1 --checkpoint of environmental status (nutrients, growth factors, space etc) G1<-->G0- resting state (quiescence, senescence, differentiated) b. S- DNA synthesis c. G2- gap phase 2 --checkpoint for genomic status (genome replicated, no mutations) |
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Cell cycle checkpoints |
1. start check point* -G1-S: is environment favorable? -->enter cell cycle and proceed to S phase 2. Intra-S checkpoint: stop DNA rep if something goes wrong 3. G2-M checkpoint: -is all DNA replicated? is environment favorable? -->enter mitosis 4. Metaphase-Anaphase transition* -are all chromosomes attached to spindle? --> trigger anaphase and proceed to cytokinesis **cancer mutations often in checkpoint proteins |
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cell cycle progression: CDK/cyclins |
Cyclin dependent kinase: -phophsorylates target proteins to change activity: 1. inactivate proteins that inhibit cell cycle (RB) 2. activate/modify proteins needed for cycle progressions (replication pretins, lamins) -present throughout cell cycle -cyclin must bind to be active Cyclin: -activate CDK -levels cycle |
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CDK/Cyclin regulation of G1 to S |
-RB-tumor suppressor blocks G1-S transition -cyclin D & E bind CDK --> phosphorylates RB --> RB inactive --> transcription factors released --> enters S |
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RB |
mutation--> proceeds into S phase prematurely --> tumor development |
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cyclin D mutation (loss of nuclear export factor) |
-too much cyclin D in nucleus --> prematurely phosphorylates in G1/phosphorylates things that shouldn't be in S phase --> tumor growth |
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CDK/cyclin regulation of S phase |
cyclin D and E: -synthesis of replication proteins -assembly of replisome -replication initiation (origin firing) cyclin A: -continued replication throughout S *replication doesn't occur w/o proper CDK phosph. |
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CDK/cyclins important for.. |
-proliferation (embryogenesis, regeneration) -development/differentiation -diseases (cancer, wasting syndrome, aging) |
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CDK/cyclin regulation of G2 to M |
CDK2/cyclin B -lamin proteins phosphorylated --> rapid disassembly of nuclear lamina |
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CDK/cyclin regulation of M to G1 |
cyclin B destroyed -lamins dephosphorylated by phosphatases --> reassembly of nuclear lamina (and associated nuclear membrane) around newly segregated chromosomes |
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Mitosis |
Interphase: -chromosomes not discernible -nuclear envelope presnet -G1, S, G2 microscopically similar Prophase: -chromosomes become discernible -nuclear envelope breaks down Metaphase: -chromosomes align on metaphase plate -mitotic spindle apparent Anaphase: -sister chromatids separate (pulled along spindle) Telophase: -chromatids in separate poles -Cytokinesis begins= cleavage furrow apparent, actin-driven Cytokinesis completion: -nuclear envelope reforms -chromosomes decondense |
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Meiosis |
2N S phase replication --> 4N Division I --> homologous chromosomes separate --> 2x 2n cells Division II --> sister chromatids separate --> 4x 1n cells |
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Karyotype |
-chromosome complement of a cell -usually visualized as a spread of mitotic chromosomes -individual chromosomes can only be visualized during mitosis -analyzed for chromosome number, malformations |
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Karyotype procedure |
1. (amniotic/clood) cells stimulated to undergo cell division, Colchicine added--> inhibited at metaphase 2. fixed 3. dropped onto glass slide 4. mitotic cells pop--> chromosomes released; interphase cells unaffected |
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normal karyotype |
22 autosomes 2 sex chromosomes |
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abnormal karyotype |
monosomy trisomy -changes in chromosome structure: (often assoc w/ cancer) --deletions --transolcations --amplifications |
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ID chromosomes from Karyotype |
1. size 2. centromere position -metacentric -submetacentric -arocentric -telocentric 3. G-banding pattern (Giemsa staining) p arm: short, q arm: long |
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Fluorescence in situ hybridization (FISH) |
-detect chromosome from DNA sequence -label specific DNA sequence and hybridize to mitotic spread --> target gene fluoresces |
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chromosome painting/spectral karyotyping (SKY) |
-visualize entire chromosome/entire karyotype via FISH -each chromosome labeled w/ different fluorphore -allows automated karyotyping -easier ID of cancer genomes (color mismatch) |
